The introduction of foreign DNA into eukaryotic host cells can serve many purposes. For example, this technique can provide a means of genetic complementation for identifying specific genes, e.g., a gene expressing an enzyme critical to a metabolic pathway can be identified by virtue of its ability to rescue cells defective in that pathway. Also, exogenous genes can be introduced for the purpose of exposing a recipient cell to a high dose of a protein not normally native to that cell, as for example, a cytotoxic protein introduced into a malignant cell for the purpose of killing it. Alternatively, foreign genes may be introduced into host cells to obtain the protein product of the foreign gene in sufficiently large amounts so that the protein can be harvested for further study or used as a pharmaceutical. In addition, the introduction of foreign genes is viewed as a promising avenue for somatic gene therapy. The goal of gene therapy is to cure inborn genetic defects by providing patients with a working copy of a missing or defective gene, or alternatively, to provide a therapeutic foreign gene product on a temporary basis for therapeutic purposes. One approach to somatic gene therapy is the ex vivo strategy, wherein cells are removed from the body, transgenic DNA is inserted into the cells, and the cells are then returned to the body. In another approach, cells in vivo are targeted by foreign DNA that is introduced directly into the patient. A variety of methods are available for introducing foreign genes into living cells.
Transfection protocols can be categorized as designed to produce "transient" or "stable" expression of the foreign gene. With currently available protocols, the line of demarcation between these two types of outcome is the integration of the introduced DNA into the host genome, and cells into which foreign DNA has become integrated are generally referred to as "stably transformed." In contrast to stable transformation, transient expression of transfected DNA does not depend on the integration of foreign DNA into host cell chromosomes. Although the majority of DNA applied to a cell is believed to be rapidly transported into the nucleus, in some systems expression can be detected for up to 80 hours post-transfection in the absence of any detectable integration (see, e.g., Gorman, C., DNA Cloning II, A Practical Approach; Glover, D. M., Ed., IRL Press, Oxford, pp. 143-190 (1985); Wynshaw-Boris et al., BioTechniques, 4:104-117 (1986)). No selection step is required before transient expression can be detected. However, only about 1-10% of cells that take up foreign DNA typically transcribe mRNA from unintegrated foreign genes (see, e.g., Gorman et al., Nucl. Ac. Res., 11:7631-7648 (1983)). Although the vast majority of transfected DNA in transiently transfected cells does not become incorporated into the host DNA, it does become incorporated in about 0.001-1% of these cells (Alam and Cook, Anal. Biochem., 188:245-254 (1990)). This small stably transfected fraction of cells is believed to play no significant or useful role in the foreign gene expression profile observed immediately after transfection. Protocols using viral vectors have been developed to increase the proportion of initially transfected cells that integrate the foreign DNA (Flamant et al., Int. J. Dev. Biol., 38:751-757 (1994); Bilbao et al., FASEB J., 11:624-634 (1997)).
Without a selection step, the expression of foreign genes generally disappears from cultures of transfected cells within two to three days. Typically, expression peaks in about 48 hours, and is detectable for only 24-80 hours (Gorman (1985); Wynshaw-Boris et al., (1986); Berthold, W., Dev. Biol. Stand., 83:67-79 (1994)). It is widely believed that most of the DNA taken up by transfected cells becomes rapidly catabolized by nucleases or becomes diluted by cell division (see, e.g., Gorman (1985); Guide to Eukaryotic Transfection with Cationic Lipid Reagents, Life Technologies; Bilbao et al. (1997)).
Because transient expression does not require that the target cells are actively dividing, it can be achieved in terminally differentiated cells that do not normally divide, although susceptibility to transfection varies dramatically among such cells. For example, naked DNA can be expressed over a long period of time when injected directly into mouse skeletal muscle (Wolff, et al., Science, 247, 1465-1468 (1990)). In other studies, naked DNA has been used as a vaccine (e.g., Cohen, J., Science, 259, 1691-1692 (1993)), and defective retrovirus vectors have been used to harness myoblasts as vehicles for delivering transgenic products (Partridge and Davies, Brit. Med. Bull., 51:123-137 (1995)).
Many studies have focused on the liposomal delivery of foreign DNA in vivo to hepatocytes (see, e.g., Wu and Wu, J. Biol. Chem. 263:14621-14624 (1988); Chow et al., J. Pharmacol. Exp. Ther., 248:506-13 (1989); Wu et al., J. Biol. Chem., 264:16985-16987 (1989); Kaneda et al., J. Biol. Chem., 264:12126-12129 (1980a); Kaneda et al., Science, 243:375-378 (1989b); Wilson et al., J. Biol. Chem., 267:963-967 (1992a); Wilson et al., J. Biol. Chem., 267:11483-11489 (1992b); Chowdhury et al., J. Biol. Chem., 268:11265-11271 (1993); Perales et al., Proc. Natl. Acad. Sci. USA, 91:4086-4090 (1994); Kormis and Wu, Seminars in Liver Disease, 15:257-267 (1995); Buolo et al., Mol. Marine Bid. Biotech., 5:167-174 (1996)). One approach to targeting foreign DNA to specific tissues in vivo is receptor-mediated liposomal delivery (reviewed in Kormis and Wu (1995)). In applying this strategy to liver, Wu and his colleagues exploited the presence of asialoglycoprotein receptors on hepatocyte surfaces to target injected liposomes to the liver. The liposomal delivery system is characterized in a number of publications (Wu and Wu (1988); Wu et al., (1989); Wilson et al., (1992a); Wilson et al. (1992b); Chowdhury et al. (1993); Perales et al. (1994)). The asialoglycoprotein was packaged into liposomes together with DNA that had formed an electrostatic complex with polylysine. When initial efforts were successful, this group attempted to maximize the stable integration of the foreign DNA by performing partial hepatectomies in the recipient rats. As regenerating liver cells provide a higher proportion of cells in S phase than are present in normal liver, this tactic was expected to increase the proportion of liver cells into which foreign DNA could integrate. After partial hepatectomy, the transgenic protein was detectable in the blood for as long as 11 weeks post-transfection (Wu et al. (1989)). At first, these investigators believed that the injected DNA had become integrated, but later experiments revealed no detectable integrated DNA, showing instead that the preserved foreign DNA resided in the plasma membrane/endosome fraction (Wilson et al. (1992b); Chowdhury et al. (1993)). This surprising observation indicated that partial hepatectomy leads to the persistence of transgenic DNA by a mechanism that is independent of DNA synthesis per se. Others have reported strategies for improving the transfection efficiency with a liposomal delivery vehicle by varying the ratio of DNA to lipids (Buolo et al. (1996)).
Another group also has employed a targeting strategy for directing injected DNA to the liver (Kaneda et al. (1989a); Kaneda et al. (1989b)). Here, transgenic DNA was packaged in liposomes with proteins normally found in the nucleus, i.e., non-histone chromosomal proteins. They observed transport of the injected vesicles to the nuclei of liver cells, and detected measurable transgene expression for up to 7 or 8 days after injection. However, this DNA did not become integrated into the liver cell chromosomes. Others have reported the successful in vivo expression of foreign DNA following the injection of CaPO.sub.4 -DNA precipitates directly into the liver, spleen, or peritoneum (see Kaneda et al. (1989a)).
A number of reagents have been shown to increase the efficiency in vitro of stable transformation. One group has reported that by controlling the pH in the culture medium during CaPO.sub.4 mediated transfection, stable transformation efficiencies as high as 50% can be achieved (Chen and Okayama, Mol. Cell. Biol., 7:2745-2752 (1987)).
Another reagent reported to enhance the expression of transfected DNA is butyric acid or its sodium salt (Gorman et al. (1983)). After exposing cells to sodium butyrate for 12 hours, Gorman et al. observed a 2-4-fold increase in the percentage of recipient cells expressing the transgene, as well as a 25-100-fold increase in the foreign gene expression levels when an SV40 enhancer was added to the construct. When other cultures transfected in the presence of butyrate were selected for stable transformants, they observed a significant increase over controls in the percentage of transfected cells that gave rise to stable transformants. However, Palermo et al. (J. Biotech., 19:35-48 (1991)) observed that butyrate induced increased transgene expression in stable transformants whether or not it had been present during the transfection step. Indeed, many reports have documented butyrate's ability to induce the synthesis of certain proteins or to increase cell differentiation in vitro. (Boffa, et al., J. Biol. Chem., 256:9612-9621 (1981); Kruh, Mol. Cell. Biochem. 42:65-82 (1982); Chabanas, et al., J. Mol. Biol., 183:141-151 (1985); Parker, J. Biol. Chem., 261:2786-2790 (1986); Kooistra, et al., Biochem. J., 247:605-612 (1987); Kaneko, et al., Canc. Res., 50:3101-3105 (1990); Nathan, et al., Exp. Cell Res., 0:76-84 (1990); Palermo, et al. (1991); Kosaka, et al., Exp. Cell Res., 2:46-51 (1991); and Oh, et al. Biotechnol. Bioeng., 42:601-610 (1993)). Optimal concentrations of butyrate for gene induction vary from cell type to cell type, and a suitable concentration range that minimizes its cytotoxic effects must be empirically determined for each type of target cell (see, e.g., Gorman (1985); Parker et al. (1986); Oh et al. (1993)). Butyric acid (or butyrate) also has been reported to reversibly suppress the growth of cultured cells (Boffa et al. (1981)), and to enhance the antitumor action of interferon (Kruh, 1982).
The usefulness of transient expression, i.e., expression from unintegrated foreign DNA, would be greatly improved if methods and reagents were available for increasing the efficiency and duration of transgene expression in the absence of selection steps.